FAQ

Single particles

Proposal needs to include an image of particle distribution in vitreous ice and preliminary 2D class averages from sample data set. It is worth mentioning what instrument, camera, pixel size, data set size was used to generate the 2D class averages so we can accurately assess the readiness of the sample for high-resolution/high-through-put data collection.

Cellular tomography and cryoFIB-milling

Provide Atlases of prepared grids showing good ice thickness and distribution of cells.

High-resolution/high-throughput tomography

Provide Atlases showing good ice distribution and some medium mag images showing distribution of sample in holes. We recommend lacey or R1.2/1.3 grids to make the most of AFIS tomographic data collection.

cryoCLEM and in situ Fluorescent directing FIB milling

Provide evidence of sample fluorescence, preferably at cryo-temperatures for in-situ FIB milling but RT will be fine if applying for access to the Leica cryoCLEM system. Please also provide Atlases of vitrified samples showing good ice thickness and sample distribution on frozen grids.

MicroED

Provide evidence of submicron crystals.

We can load a total of 11 grids per 72 hour Krios session. We recommend users load no more that 4 pre-screened grids per sample. With AFIS and FFI, it is common for users to acquire ~20,000 images for a 48 hour single particle session and up to 200 tomograms per 24 hours for molecular tomography. Thus, it is up to the user how many samples they collect on during their session.

Please be aware of the following:

• microscopes are loaded once per 48 or 72 hour session, with a single cassette containing up to 11 grids

• microscope magnification can only be changed at the start of a session during standard working hours

• only samples entered in the User Administration System (UAS) and which have validated Experimental Risk Assessments (ERAs) can be loaded into the microscopes

• a second cassette can be loaded during sessions which are longer than 72 hours but only during standard working hours

• local contact assistance outside working hours is limited to solving microscope/software failures

Note: for cryoFIB sessions only 2 grids will be loaded per session

BAG proposals provide large amounts of time for an institution or group of users over a 2 year period

If your institute is part of a Block Allocation Group (BAG), we advise you to join that BAG if you would like time at eBIC.

Rapid proposals are project based

• successful proposals are awarded a 48 hour slot on one of the four Krios microscopes
• it is expected that rapid proposals have pre-screened grids ready to ship at application and thus can be shipped as soon as the proposal is accepted
• if you are already part of a BAG, you will not be awarded rapid time
• non-UK users should apply for rapid time through one of the EU programs such as instruct-ERIC or canSERV

Diamond run schedules

The latest Diamond run schedules are available on the Diamond home page - follow the links to the operations calendars here: Quick Links - Operations Calendar.

There are no Rapid call proposal deadlines, proposals are reviewed as quickly as possible and session time will be offered at the earliest convenience.

Last Wednesday of March and September.

Yes, but we prefer you to submit a separate BAG dedicated to use of eBIC as it makes the peer review procedure a lot easier. Note: samples for eBIC sessions need to be sent to eBIC separately from any MX samples.

There is no minimum requirement for the number of researchers within a Block Allocation Group (BAG). Applications will be assessed on the number and quality of the scientific projects contained within the proposal. The smallest BAG we currently have contains three independent groups dedicated to cryo-EM.

No, the four Krios are not equivalent:

• Krios I, II and IV are equipped with Gatan BioQuantum K3 imaging filter and detector

• Krios III is equipped with Thermo Fisher Scientific Selectris X imaging filter and Falcon 4i detector

Please see instruments page for current set up of individual microscopes or contact your local contact for information.

Krios III.

No. Diamond recommends using Globus/FTP to transfer data to your home institution. Please see our user guide for more information and options for remote access and analysis.

Please see the remote access and analysis user guide  for more details.

Block Allocation Group (BAG)

Discuss with your BAG Principal Investigator which microscope you would like to use. They can then request for time on a specific instrument during the next run allocation.

Rapids

Contact ebicadmin@diamond.ac.uk with requests and we will do our best to accommodate.

Details are listed in the eBIC web page Publications acknowledgement. The following statement should always be included in any publications "(We acknowledge) Diamond for access and support of the cryo-EM facilities at the UK national electron Bio-Imaging Centre (eBIC), proposal BI####.”